Targeting Leishmania donovani Sphingosine Kinase 1 using PF-543 enhances immune response and limits parasite load

BackgroundSphingosine-1-phosphate (S1P) is a bioactive lipid mediator regulating apoptosis, proliferation, and immune responses. While S1Ps presence in Leishmania donovani phagolysosomes has been reported, the role of sphingosine kinases, especially SphK1, in parasite survival and host immune modulation remains underexplored. This study investigates the molecular and functional role of L. donovani SphK1 (LdSphK1) and evaluates the antileishmanial potential of PF-543, a specific SphK1 inhibitor. MethodsLdSphK1 and human SphK1 (rhSphK1) were cloned, expressed in E. coli, purified, and analyzed by SDS-PAGE. Enzymatic activity and inhibition by PF543 were assessed using NBD-S1P-based fluorometric assays. Protein-ligand interactions were analyzed using Microscale Thermophoresis (MST). Leishmania promastigotes overexpressing LdSphK1 were studied via confocal microscopy, and their viability and infectivity were assessed in vitro. THP-1 macrophages infected with L. donovani were treated with PF543 alone or with Amphotericin B and analyzed by MTT assay, RT-PCR, Giemsa staining, ELISA and immunoblotting. In vivo efficacy was tested in L. donovani-infected Swiss mice. ResultsrLdSphK1 ([~]102 kDa) and rhSphK1 ([~]50 kDa) were enzymatically active and significantly inhibited by PF-543. MST confirmed high-affinity binding of PF-543 (KD [~]29 microMolar). In L. donovani SphK1 overexpressor (LdSphKa) promastigotes, PF543 inhibited SphK1 activity and reduced parasite infectivity, more than in wildtype L. donovani promastigotes. Notably, PF543 treatment reduced parasite infectivity in vitro, lowered amastigote load by [~]40%, and promoted a pro-inflammatory cytokine shift ({uparrow}IL-12, {uparrow}TNF-, {downarrow}IL-10). Inhibition of ceramide synthesis and S1P supplementation revealed that S1P rescues ceramide-induced parasite death, implicating SphK1 in parasite survival. PF543 and Amphotericin B demonstrated synergistic anti-parasitic effects both in vitro and in vivo, with >90% reduction in parasite burden in mice. ConclusionPF543 is a potent inhibitor of SphK1, impairing parasite survival and modulating host immune responses. When combined with Amphotericin B, it offers a synergistic therapeutic strategy against visceral leishmaniasis, warranting further clinical exploration. Author SummaryLeishmaniasis, a neglected tropical disease, has limited available treatments and is becoming more resistant to medications. In this study, we explored the therapeutic potential of PF-543, a potent sphingosine kinase 1 (SphK1) inhibitor (demonstrated anticancer effects in various preclinical models) against Leishmania donovani. We successfully cloned and purified both Leishmania and human SphK1 proteins and confirmed PF-543 binding through biochemical and biophysical assays. Overexpression of LdSphK1 in parasites enhanced their survival and infectivity. In vitro, PF-543 treatment of infected macrophages decreased amastigote burden, shifted cytokine profiles towards a pro-inflammatory state, and enhanced host cell apoptosis. Notably, PF-543 acted synergistically with Amphotericin B, the current clinical drug, both in vitro and in vivo in Swiss mice, drastically lowering parasite burden. This study highlights the possibility of combination therapy and finds PF-543 to be a promising irresistible host-targeted antileishmanial drug. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/652326v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1926575org.highwire.dtl.DTLVardef@4305f4org.highwire.dtl.DTLVardef@3c10c7org.highwire.dtl.DTLVardef@171801e_HPS_FORMAT_FIGEXP M_FIG C_FIG